Thyroxine (T4), the principal circulating thyroid hormone (TH), is widely believed to be a prohormone that must be converted by the 5'-deiodinases (D1 and D2) to 3,5,3'-triiodothyronine (T3). T3 in turn binds to nuclear TH receptors (TRs) to mediate the genomic effects of this class of hormones. THs modulate numerous processes in almost every tissue in the body throughout life, and have long been known to play an essential role in brain development. TH deficiency in humans during the fetal and neonatal period results in irreversible developmental abnormalities including mental retardation, deafness and ataxia. Analogous impairments result from perinatal hypothyroidism in rodents. While the differential expression of TRs in the developing brain is a key element in determining the extent of TH action, the availability of the appropriate amount of their ligand is equally important. In te developing brain TH action is thought to be mediated predominantly, if not exclusively, by T3, with the majority of this ligand generated within the cells of this organ from T4 by the D2. However, this model cannot account for several recent findings. First, mice deficient in the D2 (the D2KO and D1/D2KO) and therefore totally dependent on the serum as the source of T3 in neural tissues, fail to show the severe neurobehavioral phenotype expected if D2 were the critical source of brain T3 during development. Second, a mouse deficient in the MCT8, a protein that preferentially transports T3 from serum into the brain, also demonstrates minimal neurological abnormalities. It is notable that in both these transgenic mouse strains the brain T3 content during the developmental period is reduced 40-50%, a reduction comparable to the level seen in highly compromised hypothyroid mice. Third, our preliminary observations indicate that a mouse deficient in both 5'- deiodinases and the MCT8 (the D1/D2/MCT8KO) also fails to manifest the severe defects in locomotion, learning and memory seen in the hypothyroid mouse, despite a brain T3 content significantly less than that observed in the latter condition. However, compared with WT mice, the serum and brain T4 contents are elevated in the D1/D2KO and D1/D2/MCT8KO mice and are substantial in the MCT8KO mouse. In sharp contrast, the T4 level is exceedingly low in the hypothyroid mouse, raising the possibility that T4 is directly effecting brain development. These observations lead us to hypothesize that T4 per se has intrinsic physiological activity in the brain that contributes significantly to the coordinated effcts of TH during development. We plan to test this hypothesis using our newly created D1/D2KO/Pax8-/- mouse, which can neither synthesize TH, nor convert administered T4 to T3. This unique model will allow for the first time a determination of the independent effects of T4 and T3 during development in vivo. These findings may result in a paradigmatic shift in our understanding of TH action in general and the importance of maternal T4 during pregnancy in particular.